Here, we demonstrate for the first time that the BY-2 cell-free lysate also allows the efficient production of several classes of secondary metabolites. As case studies, we synthesized lycopene, indigoidine, betanin, and betaxanthins, which are useful in the food, cosmetic, textile, and pharmaceutical industries. Production was achieved by the co-expression of up to three metabolic enzymes. For all four products, we achieved medium to high yields. However, the yield of betanin (555 μg/mL) was outstanding, exceeding the level reported in yeast cells by a factor of more than 30. Our results show that the BY-2 cell-free lysate is suitable not only for the verification and optimization of metabolic pathways, but also for the efficient production of small to medium quantities of secondary metabolites.Cell-free expression systems enable the production of proteins and metabolites within a few hours or days. Removing the cellular context while maintaining the protein biosynthesis apparatus provides an open system that allows metabolic pathways to be installed and optimized by expressing different numbers and combinations of enzymes. This facilitates the synthesis of secondary metabolites that are difficult to produce in cell-based systems because they are toxic to the host cell or immediately converted into downstream products. Recently, we developed a cell-free lysate derived from tobacco BY-2 cell suspension cultures for the production of recombinant proteins. This system is remarkably productive, achieving yields of up to 3 mg/mL in a one-pot  They are generally not required for normal growth and development and instead facilitate ecological interactions, although the benefit to the producer is not always clear. However, many secondary metabolites, including alkaloids, terpenoids, polyketides, antibiotics, peptides, and growth hormones, have structures or biological activities that make them useful for applications in the chemical, agri-food, cosmetic, and pharmaceutical industries. Traditionally, secondary metabolites are extracted from their natural sources using solvents or are produced by chemical synthesis. However, both methods tend to be inefficient and harmful to the environment, making them costly and unsustainable. Furthermore, many natural products are too complex for chemical synthesis. To overcome these drawbacks, the industrial-scale production of secondary metabolites is often achieved by the metabolic engineering of microbial cells. However, the preparation of production strains is laborious and the heterologous production of some secondary metabolites can be difficult or even impossible due to the lack of precursors, the low activity of the introduced enzymes, or the inhibition of cell growth by the product  produced and excreted by some species of bacteria. It is a blue pigment that has been considered as a more sustainable alternative for industrial dyes , but it nsferase .L-3,4-dihydroxyphenylalanine (L-DOPA) by a cytochrome P450 hydroxylase and then to betalamic acid by a DOPA 4,5-dioxygenase (DOD) enzyme. Betalamic acid then reacts with various amino acids or amines to form diverse yellow-orange betaxanthins. Alternatively, L-DOPA is oxidized by the diphenolase activity of a cytochrome P450 to dopaquinone, which spontaneously cyclizes into cyclo-DOPA. The latter is converted to cyclo-DOPA-5-O-glucoside by a glucosyltransferase and reacts with betalamic acid to form the red pigment betanin , NRPs (indigoidine), and alkaloid-like betalains (betanin and betaxanthins). Given the inherent color of the products, their accumulation in the cell-free system can easily be monitored using non-invasive methods and the performance of individual enzymes can be determined by separate expression and step-by-step construction of the pathway. In addition, precursors can be added to the cell-free system to maximize product accumulation. This is the first step toward the wider use of the BYL platform for the engineering of metabolic pathways to produce valuable secondary metabolites.lacZ sequence and the F1 origin of replication were removed. To construct the control vector pLenEx_Strep-eYFP, expressing the fluorescent protein eYFP including an N-terminal streptavidin affinity tag, the Strep-eYFP sequence was amplified by PCR using primers 5′-GGT AGT CCA TGG CTT GGT CTC ATC CGC AAT TC-3′ and 5′-GAC ACG GGT ACC TTA TTA CTT GTA CAG CTC GTC CAT GC-3′, with pIVEX_GAA_Omega_Strep-eYFP as the template (NcoI and KpnI (underlined in the primer sequences) and inserted at the NcoI and KpnI sites of pLenEx.All cDNAs were transferred to the expression vector pLenEx , which wtemplate . The PCRPantoea ananatis CrtE, CrtB, and CrtI proteins  for lycopene synthesis, the Streptomyces lavendulae BpsA and Bacillus subtilis Sfp proteins  for indigoidine synthesis, and the Beta vulgaris CYP76AD1_W13L, Mirabilis jalapa DOD, M. jalapa cDOPA5GT and B. vulgaris CYP76AD5 proteins  for betalain synthesis, were synthesized as double-stranded DNA fragments  including overhangs suitable for Gibson assembly , replacing the Strep-eYFP cDNA with the sequence encoding the enzyme of interest (NcoI restriction site. The bpsA and sfp sequences were codon optimized for tobacco (Nicotiana tabacum) using the codon optimization tool provided by Integrated DNA Technologies, and the first codon after the start codon was replaced with glycine to create an NcoI restriction site as previously described  in a 50-mL polypropylene tube . After centrifugation at 6,800 × g for 1 h at 25°C in a swing-bucket rotor, evacuolated protoplasts were recovered from the 40–70% (v/v) Percoll solution interface and suspended in three volumes of TR buffer (30 mM HEPES-KOH (pH 7.4), 60 mM potassium glutamate, 0.5 mM magnesium glutamate, 2 mM DTT) supplemented with one tablet per 50 mL of Complete EDTA-free Protease Inhibitor Mixture . The protoplasts were disrupted on ice using 15 strokes of a Dounce homogenizer , and the nuclei and non-disrupted cells were removed by centrifugation at 500 × g for 10 min at 4°C. Standard cell-free reactions contained 40% (v/v) BYL, 20 mM HEPES-KOH (pH 7.8), 9 mM magnesium glutamate, 20 mM potassium glutamate, 4 mM ATP, 1.6 mM GTP, 1.6 mM CTP, 1.6 mM UTP, 100 μg/mL chloramphenicol, and 30 ng/μL in-house T7 RNA polymerase. The reaction mix was supplemented with 2.5% (w/v) trehalose as a cryoprotectant and then frozen in 1-mL aliquots at –80°C.The tobacco cell-free lysate (BYL) was prepared as previously described  with minCoupled transcription–translation reactions were carried out in aliquots of 50, 150, or 500 μL at 25°C and 70% humidity, shaking at 500 rpm (12.5 mm amplitude) in a Lab-Therm LT-X incubator shaker . Ten to 30 ng/μL (5–10 nM) plasmid DNA was added to the cell-free reaction mix. BYL mixtures for betanin and indigoidine production were optionally supplemented at the start of the reaction with 5 mM tyrosine and 2.5 or 5 mM glutamine, respectively, as precursors.Samples were separated by SDS-PAGE in precast NuPAGE 4–12% (w/v) polyacrylamide Bis-Tris gels  alongside PageRuler Prestained Protein Ladder markers (Thermo Fisher Scientific). The gels were stained with Coomassie Brilliant Blue R-250.g for 30 s. The upper organic phase was transferred to a fresh microcentrifuge tube and the absorbance was measured at 472 nm . The samples were then analyzed by chromatography using an Ultimate3000 UHPLC system (Thermo Fisher Scientific) including a quaternary pump, autosampler, column oven and UV-detector. The sampler temperature was set to 10°C and the column compartment to 25°C. Compounds of interest were analyzed in reversed phase mode on a LiChrosorb RP-18 column  . We separated 5.0-μL samples at a flow rate of 0.5 mL/min using 5.0% (v/v) methanol plus 94.9% (v/v) water with 0.1% (v/v) trifluoroacetic acid as eluent A and 95.0% (v/v) methanol plus 4.9% (v/v) water with 0.1% (v/v) trifluoroacetic acid as eluent B. The program began with an isocratic segment at 100% eluent A for 5 min, followed by a linear gradient to 60.0% eluent B (5–17 min), a linear gradient to 100% eluent B (17–19 min), an isocratic segment at 100% eluent B (19–23 min) and finally a linear gradient to 100% eluent A (23–24 min). The column was re-equilibrated for 6 min in 100% eluent A. Betanin was detected at 536 nm and was compared to a commercial betanin standard (Sigma-Aldrich). The formation of betaxanthins was detected by optical verification of a color change in the BYL reaction samples from pale yellow to bright yellow to orange.bpsA and sfp sequences were codon optimized for tobacco to facilitate comparison with a previous study in which codon-optimized sequences were used for cell-based production in Rhodosporidium toruloides  to ensure efficient cDNA synthesis. For similar reasons, a C-terminal StrepII tag sequence was introduced into the CrtE, CrtB, and CrtI constructs since All target cDNAs were cloned separately into the expression vector pLenEx for the cell-free production of lycopene (three genes), indigoidine (two genes), and betalains (four genes) . Codon oruloides . In the in vitro transcription and translation, such as nucleoside triphosphates (NTPs), amino acids and salts, are already provided in the lysate. Accordingly, only vectors carrying the appropriate cDNAs need to be added to the cell-free lysate to facilitate rapid protein biosynthesis, enzyme analysis and the accumulation of secondary metabolites in a single reaction vessel.All 10 target genes were expressed under the control of the T7 promoter in the pLenEx expression vector. The T7 polymerase and all other factors necessary for P. ananatis. These enzymes convert IPP and its isomer DMAPP to lycopene in three steps  in the individual reactions. In the combinatorial reactions we used equimolar concentrations of each construct and a total molar concentration of 5 nM. After 21 h, the reaction with three plasmids producing the full complement of enzymes  showed the most intense orange color, indicating the accumulation of large amounts of lycopene . In contS. lavendulae blue pigment synthetase A (bpsA) and B. subtilis 4′-phosphopantetheinyl transferase (sfp) genes. Following the activation of BpsA by the Sfp transferase, BpsA catalyzes the condensation of two molecules of L-glutamine to form indigoidine . Indigoidine formation was detected optically based on a color change from pale yellow to blue-green. As expected, the expression of Sfp alone did not change the color of the lysate  the red-violet betacyanin betanin, which requires the monophenolase as well the diphenolase activity of the cytochrome P450 BvCYP76AD1_W13L from B. vulgaris, MjDOD, and the glucosyltransferase MjcDOPA5GT from M. jalapa  yellow-orange betaxanthins, which requires the monophenolase activity of the cytochrome P450 BvCYP76AD5 from . jalapa . As abov. jalapa . As antiL-DOPA was channeled toward the synthesis of betalamic acid and subsequently betaxanthins . The exclusive monophenolase activity of BvCYP76AD5 ensured that the xanthins . The reaxanthins . The reaxanthins , indicatxanthins . The co-xanthins . Adding L-DOPA but the additional diphenolase activity converted it into cyclo-DOPA . In this reaction, the monophenolase activity of BvCYP76AD1_W13L produced clo-DOPA . When alclo-DOPA . Adding clo-DOPA . Once agTo determine the betanin content, the cell-free reactions were repeated at the 500-μL scale using the same molar concentrations of template DNA (1.73 nm each) with and without 5 mM tyrosine. The betanin concentration was determined after 24 h by UHPLC. The yield in the absence of tyrosine was 125 μg betanin per mL cell-free reaction, whereas the provision of 5 mM tyrosine increased the yield to 470 μg per mL. Higher molar concentrations of template DNA (3.33 nm each) increased the betanin yield even further to 555 μg per mL cell-free reaction. These remarkable yields demonstrate that the cell-free BYL system is not only suitable for the analysis of metabolic pathways but also for the rapid production of target molecules.in vitro transcription–translation in batch reactions, which is ∼15 times more productive than other eukaryotic cell-free systems in batch mode  can be added to the thawed lysate to initiate biosynthesis. The cell-free platform thus provides a flexible, on-demand tool for the analysis and optimization of metabolic pathways, and can provide sufficient quantities of secondary metabolites for various assays without the costly infrastructure required for cell-based protein production. It is therefore unsurprising that cell-free expression has already been exploited for such approaches, although these systems are typically derived from microbial cells . The reported yield was up to 250 μM (62 mg/L). More precise quantitative analysis was not possible due to the lack of suitable standards, which is also why we did not determine the concentration of indigoidine in our study. Nevertheless, the colorimetric assay confirmed the accumulation of large amounts of indigoidine, indicating that the BYL system can be harnessed for the rapid engineering and optimization of indigoidine biosynthesis.In a similar effort to improve the synthesis of indogoidine, the ectively . Indigois system . This sytrations . In contE. coli, with yields of up to 288 mg per L (S. cerevisiae, achieving a titer of 17 μg per mL for betanin, the most industrially relevant betalain. To our knowledge, the cell-free biosynthesis of betalains has not been reported thus far. The successful production of betaxanthins and betacyanins in the BYL system is therefore the first step toward the engineering and optimization of betalain biosynthesis in this platform. Notably, we achieved yields of up to 555 μg betanin per mL BYL solution, which is 32 times higher than previously achieved by cell-based expression in S. cerevisiae (The production of betalains has also been transferred to microbes by metabolic engineering. Various betaxanthins and betacyanins have been produced in mg per L . Grewal revisiae  and thusWe have shown that the cell-free BYL system can support the production of secondary metabolites, using representatives from the carotenoids, NRPs and betalains as case studies. In the future, we will optimize the BYL system to improve the accumulation of lycopene, indigoidine and betalains by testing enzymes from alternative organisms and/or by optimizing enzyme sequences and improving reaction conditions before transferring optimized pathways to cell-based production platforms. In addition, metabolic pathways can be modified and augmented to synthesize products with improved or new properties, including xenobiotics that are not found in nature because they are unstable and/or quickly metabolized in living cells. This could provide a new source of novel active substances as drug leads. The cell-free BYL system can be scaled up inexpensively by using more BY-2 cell biomass for lysate preparation, with work underway to reach scales of 1 L and even 10 L per reaction . This woThe original contributions presented in the study are included in the article/MB and SS conceived the study and wrote the manuscript. MB, NH, and AC conducted the experiments and collected the data. All authors interpreted data, proofread, and approved the manuscript.SS is member of the Scientific Advisory Board of LenioBio GmbH, which distributes the BY-2 cell-free lysate developed by Fraunhofer IME and Dow AgroSciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Melissococcus plutonius is a major bacterial disease of honey bees. Strains of the causative agent exhibit genetic heterogeneity, and the degree of virulence varies among strains. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. However, the barrier is degraded during the course of infection, and M. plutonius cells eventually directly interact with the midgut epithelium. As M. plutonius possesses genes encoding putative PM-degrading proteins , we constructed PM-degrading protein gene-knockout mutants from a highly virulent M. plutonius strain and investigated their role in the pathogenesis of EFB. In larvae infected with the triple-knockout mutant, which has no PM-degrading protein genes, M. plutonius that proliferated in the larval midguts was confined to the sac of the PM. However, the midgut epithelial cells degenerated over time, and the mutant killed approximately 70–80% of bee brood, suggesting that although the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain.European foulbrood (EFB) caused by  Apis mellifera L.) is the most important commercial pollinator aiding in the production of a variety of agricultural and horticultural crops, diseases of the western honey bees have a great economic impact worldwide. European foulbrood (EFB) caused by a Gram-positive lanceolate coccus Melissococcus plutonius1 is one of the globally distributed and economically important infectious diseases of honey bees3. EFB affects mainly young larvae and usually kills them 1–2 days before being sealed in their cells4. However, the degree of virulence varies among M. plutonius strains, presumably among genotypes. M. plutonius strains can be grouped into three clonal complexes  by the multilocus sequence typing analysis (https://pubmlst.org/mplutonius/)8. In previous in vitro experimental infections of A. mellifera larvae9, the representative CC12 strain (DAT561) was extremely virulent and killed all bees before pupation, whereas the representative CC3 strain (DAT606) was less virulent than the CC12 strain, and a part of the infected larvae pupated. In contrast, the representative CC13 strain (DAT585) had negligible effects on the mortality of bee larvae9. Lewkowski and Erler10 also showed virulence differences among M. plutonius strains with different genetic backgrounds. In their study, a European CC3 strain (49.3 from Switzerland) had consistently higher virulence against honey bee larvae than European CC13 strains (119 from Switzerland and 4–127 from Sweden). More recently, Grossar et al.11 quantified the virulence of 16 CC3 and CC13 isolates from five European countries using a standardized in vitro assay. Although the degree of virulence was independent of the sequence types of the tested M. plutonius isolates in the study, virulence varied greatly among the 16 isolates11. However, the genetic basis of the distinct pathological characteristics of different M. plutonius strains has not been fully elucidated.As the western honey bee  and moderately virulent strain DAT606 (CC3) possess pMP19, whereas the avirulent strain DAT585 (CC13) does not have the plasmid16. As the loss of pMP19 from CC3 strains resulted in a significant decrease in the virulence of the strains, pMP19 was considered to be a virulence determinant at least for CC3 strains16. However, irrespective of the presence of pMP19, the CC12 strain DAT561 was virulent and killed more than 80% of the honey bee brood in our in vitro infection model16, strongly suggesting the presence of unidentified pathomechanisms functioning in the virulence of CC12 strains.In a recent study, Djukic et al.Paenibacillus larvae. Ingested spores germinate in the larval midgut and vegetative P. larvae cells proliferate within the midgut18. In the larval midgut, the peritrophic matrix (PM), a chitin and glycoprotein layer that covers the lumen side of the midgut epithelium, protects the midgut epithelium from damage by pathogens19; however, P. larvae degrades this protective layer by the chitin-binding and -degrading protein PlCBP49, breaches the gut epithelium, invades the hemocoel and proliferates in the paracellular space21. The infected larvae finally die from septicemia24. In the absence of PlCBP49 expression, PM degradation was markedly reduced and P. larvae virulence was nearly abolished; therefore, the PM-degrading protein PlCBP49 is considered a key virulence factor in P. larvae21.In American foulbrood (AFB), another contagious bacterial infectious disease of honey bee brood, the infectious cycle begins when a larva eats spores of the causative agent M. plutonius is also known to colonize the larval midgut. However, the impact on the midgut differs depending on the strain ingested. When A. mellifera larvae were experimentally infected with the highly virulent strain DAT561, orally ingested bacterial cells were localized on the surface of the PM. The DAT561 cells then rapidly multiplied on the PM and eventually almost completely occupied the midgut lumen25. Similar to AFB, the PM was degraded during the course of DAT561 infection and M. plutonius directly interacted with the midgut epithelium25. In the infected larvae, degeneration of the epithelial cells was also observed25. In contrast, when A. mellifera larvae were infected with a low virulent strain of CC13 , the intestinal wall was not destroyed, and the PM and intestinal epithelium remained intact during the 15-day experimental period26. Therefore, as with AFB by P. larvae, PM-degrading proteins may be indispensable in the pathogenesis of EFB by the highly virulent M. plutonius strain DAT561 of CC12.M. plutonius strains, Djukic et al.12 found three putative PM-degrading protein genes in the genomes of M. plutonius. One of the genes encodes the chitin-binding domain-containing protein (Cbp), which exhibited 37% amino acid sequence similarity to PlCBP49 of P. larvae 12. Enhancins, which belong to a class of metalloproteases originally found in some baculoviruses, are known to promote viral infection by degrading the PM of the insect midgut27, whereas endo-α-GalNAc-ase is the enzyme that catalyzes the hydrolysis of the O-glycosidic bond between α-GalNAc at the reducing end of mucin-type sugar chains (O-glycan) and serine/threonine residues of proteins28. Among the three putative PM-degrading proteins, the expression of Efp and endo-α-GalNAc-ase in naturally infected larvae has been confirmed12. However, it is unknown whether these putative PM-degrading proteins affect the integrity of the PM of honey bee larvae and function as key virulence factors of M. plutonius. As our research group recently determined the genome sequences of the highly virulent M. plutonius strain DAT561, moderately virulent strain DAT606, and avirulent strains DAT585 and ATCC 35311 29, in order to investigate the role of the putative PM-degrading proteins in the pathogenesis of EFB, in particular that by the highly virulent strain DAT561, we compared amino acid sequences of the proteins among the strains. In addition, we constructed a series of putative PM-degrading protein gene-deficient mutants from DAT561 and investigated their characteristics using the in vitro A. mellifera larvae infection model.In a recent comparative genome analysis of M. plutonius has a putative signal peptide and two conserved domains  , whereas Mucin_bdg is the putative binding domain for the substrates of enhancin and other similar metallopeptidases (https://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=pfam03272). In the previous genome analysis of M. plutonius12, Efp was found to be conserved in all European strains analyzed , which is also annotated as the module belonging to the auxiliary activities 10 (AA10) family or chitin binding module 33 (CBM33) family of lytic polysaccharide monooxygenases21  and does not have other domains conserved in PlCBP49  between DAT561 of CC12 and DAT606 of CC3, and their amino acid sequences were 96.2% identical  . Moreover, in CC13 strains, the enzyme was truncated due to nonsense mutations at the C-terminal region, resulting in deletion of the GalBD_like domain ; therefore, endo-α-GalNAc-ase of CC13 strains may not be functional. Indeed, the eng gene of CC13 strains was annotated as pseudo in the GenBank database  were considered to be pseudogenes in the avirulent strain DAT585, and a 90-bp in-frame deletion was found in the eng gene in the moderately virulent strain DAT606. In contrast, in the highly virulent strain DAT561, all three genes were considered to be intact and they may therefore be involved in the high virulence of the CC12 strain. However, the true roles of these proteins in the pathogenesis of EFB cannot be determined by only in silico analyses. In addition to evaluation of the in silico data, it is essential to analyze the genes using isogenic mutants.In summary, two of the three putative PM-degrading protein genes  cleaves chitobiose to N-acetylglucosamine (GlcNAc), and GlcNAc is taken up by the bacterium via the GlcNAc-specific phosphotransferase sugar transporter31. However, in the genomes of M. plutonius, no EfChi18A homologue was found by the blastp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi).In order to clarify the role of the three putative PM-degrading proteins in the pathogenesis of EFB caused by the highly virulent strain DAT561, we constructed single-, double- and triple-knockout mutants of the 61 Table  as descrted Fig. . The parted Fig. 14. Althones Fig. . Deletio A. mellifera larvae infection model as described in Methods and Supplementary Tables 16.The virulence of the PM-degrading protein gene deletion mutants was evaluated using the in vitro16. As mentioned above, we expected the three putative PM-degrading proteins to be key virulence factors in the highly virulent M. plutonius strain. However, all constructed mutants were still virulent in honey bee broods. Even in the triple-knockout mutant (DAT561ΔpMP19-efp-cbp-eng)-infected group, the survival rate at day 21 pg was only 20.8%, almost the same level as in the DAT561ΔpMP19-infected group , there was no significant difference in survival between each mutant and DAT561ΔpMP19 . As reported in the previous study by Lewkowski and Erler10, different results may be obtained when queens with different genetic backgrounds are used for in vitro assays. However, in this experiment, larvae from two different queens were used (Supplementary Table M. plutonius in inocula slightly differed from strain to strain (1.1–1.5 × 106 CFU/ml), the inoculation dose of DAT561ΔpMP19-efp-cbp-eng  was the same as that of DAT561ΔpMP19  was slightly higher than that of the DAT561ΔpMP19-infected group, there was no significant difference in survival between the two groups . For histopathological analysis, we sampled only larvae that survived to the day of sampling  compared with non-infected control larvae (the average weight at day 4 pi was 147.55 mg).In contrast, all of the DAT561ΔpMP19-infected larvae had immunohistochemical (IHC) staining-positive gut Fig. b. At daylls Fig. b. In lar PM Fig. b. In thells Fig. b. In addM. plutonius cells were also detected in the midgut of all the DAT561ΔpMP19-efp-cbp-eng-infected larvae, suggesting rapid proliferation of ingested M. plutonius in larvae , demonstrating that disturbed PM integrity is sufficient to cause larval mortality even in the absence of a histologically visible effect on PM formation20. In the DAT561ΔpMP19-efp-cbp-eng-infected larvae, disturbance of PM integrity and/or PM formation by unidentified M. plutonius factors may have affected the normal larval development and survival.So, why did the DAT561ΔpMP19-efp-cbp-eng-infected larvae, degenerated or disintegrated epithelial cells were also observed at day 4 pi  may have affected the integrity of the midgut epithelial cells. Previous genome analysis by Djukic et al.12 identified an Enterococcus-type tyrosine decarboxylase gene cluster, which is involved in tyramine production. Kanbar et al.39 reported that tyramine production of E. faecalis has highly toxic effects on honey bee larvae leading to classical EFB-like symptoms. Therefore, tyramine produced by DAT561ΔpMP19-efp-cbp-eng may be one of the factors that permeates the PM and damages the midgut epithelial cells. Of note, the genes encoding tyrosine decarboxylase are putatively non-functional due to a nonsense mutation in the type strain of M. plutonius 12, which did not destroy the intestinal epithelium in infected larvae26. These reports suggest the importance of tyramine production in EFB.In most of the DAT561ΔpMP19-M. plutonius are involved in degradation of the PM in honey bee larvae. As larval mortality is known to increase by only disturbing the PM integrity20, the putative PM-degrading proteins, which can actually degrade the PM, probably function as virulence factors of M. plutonius. However, as deletion of the PM-degrading protein genes did not decrease larval mortality by the highly virulent CC12 strain, neither of the three proteins was considered as the indispensable virulence factor in the CC12 strain at least in the in vitro A. mellifera larvae infection model. However, these proteins may interact with other genes/proteins or are part of a cascade that promotes pathogenesis and the distribution of EFB under colony conditions. In this study, although we focused on only the pathomechanisms of EFB caused by the virulent CC12 strain, the reason why some CC13 strains including DAT585 and LMG 20360 are low virulent is also interesting. As strain LMG 20360 lost the ability to degrade the PM26, the virulence of CC13 strains may increase by repairing mutations in the PM-degrading protein genes. However, to verify these hypotheses and elucidate the pathogenesis of EFB caused by different M. plutonius strains, further studies of the role of the putative PM-degrading proteins at the colony level and identification of other virulence factors will be necessary.This study demonstrated that all or some of the three putative PM-degrading proteins of M. plutonius strains12, their roles in the pathogenesis of EFB were unknown due to a lack of data from isogenic mutants of the genes. In this study, we constructed a series of putative PM-degrading protein gene knockout mutants from the highly virulent CC12 strain DAT561, and demonstrated that M. plutonius DAT561 can kill honey bee larvae even in the absence of the three genes. As the plasmid pMP19 plays a significant role in the virulence of M. plutonius strains only in CC316, we cannot exclude the possibility that the three putative PM-degrading proteins are also essential for M. plutonius strains with specific genotypes to kill honey bee larvae aggressively; however, our present study suggested that the three proteins were dispensable virulence factors at least in M. plutonius DAT561, and demonstrated the usefulness and importance of our genetic tools and techniques42 for evaluating the roles of M. plutonius genes in the pathogenesis of EFB. In this study, we were unable to find indispensable virulence factors in the highly virulent M. plutonius strain; however, the existence of virulence factors that can go through the PM and affect the integrity of the PM and/or midgut epithelial cells in honey bee larvae was suggested. The previous comparative genomic analysis12 detected many other virulence factor candidates, including bacteriocins, bacteria cell surface- and host cell adhesion-associated proteins, an enterococcal polysaccharide antigen, an enzyme involved in tyramine production and capsule-associated proteins. We are currently performing experiments to elucidate the role of these putative virulence factors using genetic tools and techniques for M. plutonius, in addition to the in vitro A. mellifera larvae infection model.The rapid advances in genome sequencing technologies are providing vast quantities of nucleotide sequence data and many putative virulence factors have been found in many pathogens. However, the true roles of the factors in the pathogenesis of infectious diseases cannot be determined by only in silico analyses, and it is essential to analyze live bacteria, including isogenic mutants, in addition to evaluating in silico data. Indeed, although the genes encoding Efp, Cbp and endo-α-GalNAc-ase were described as putative virulence factors in the previous comparative genomic study of Melissococcus plutonius strains used in this study are listed in Table lacZ´ gene40, was used to construct gene knockout vectors for gene manipulation of M. plutonius strains. E. coli TOP10  was used to host the constructed vectors. M. plutonius and E. coli strains were cultured using KSBHI broth or agar33 at 34.5–35 °C under anaerobic conditions and LB  broth or agar at 37 °C under aerobic conditions, respectively, as described previously16. For maintenance of the plasmid vectors, chloramphenicol was added to the media at the following concentrations: 8–15 µg/ml for E. coli and 4–15 µg/ml for M. plutonius. For selection of E. coli strains with pSET6s-based recombinant plasmids by the blue-white screening technique, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside was added to LB plates at 100 µg/ml. Bacterial DNA was prepared using InstaGene Matrix  in accordance with the manufacturer’s instructions.M. plutonius strains with different genetic backgrounds, PlCBP49 of P. larvae ATCC 9545, CBP21 of S. marcescens and EfCBM33A of E. faecalis were retrieved from the GenBank database at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). Multiple sequence alignments of the proteins were computed using ClustalW . Signal peptides were predicted by SignalP-5.0 .Amino acid sequences of the putative PM-degrading proteins of efp, cbp and eng genes, upstream and downstream regions of the genes were amplified from genomic DNA of the M. plutonius strain DAT561 by PCR, and fused by overlap-extension PCR41 using the primers and enzymes listed in Supplementary Table 40 in E. coli TOP10 (Invitrogen). The resulting plasmids were introduced into M. plutonius DAT561 or DAT561 derivatives  by electroporation42, and double-crossover gene deletion mutants were generated according to the procedures described previously41. Deletion of the target genes was confirmed by PCR using the primers and enzymes listed in Supplementary Table For construction of precise in-frame deletions in the 9 and carbohydrate test media supplemented with four different carbon sources  (Supplementary Table 600) of 0.4 and 0.6 using an SP-300 spectrophotometer  for growth measurements in KSBHI broth and carbohydrate test media, respectively. After adding 1/40 of the volume of each adjusted bacterial suspension to the media, the cultures were incubated at 35 °C under anaerobic conditions. The OD600 of the cultures was measured at 0, 6, 9, 12, 15, 18, 21, 24, 27 and/or 30 h after incubation. Growth of each strain in KSBHI broth was measured in duplicate in each test and the tests were repeated twice, whereas growth in carbohydrate test media was measured using five independent culture tubes. To confirm acid production from supplemented carbon sources, bromocresol purple (FUJIFILM Wako) was added to the suspension and carbohydrate test media to a final concentration of 0.003% (w/v), and a color change of the media was observed after a 36-h incubation.KSBHI broth16. Briefly, M. plutonius strains cultured on KSBHI agar plates33 were suspended in sterile H2O at a final concentration of approximately 1–2 × 107 CFU/ml, and the suspension and artificial diet B'  and reared using 48-well cell culture plates until day 21 pg as described previously16. Larvae in non-infected control groups were fed normal artificial diet  according to the manufacturer’s instructions. IHC staining was also performed using commercial kits (Histofine Simple Stain MAX-PO (MULTI) and Histofine Simple Stain AEC Solution, Nichirei Biosciences Inc., Tokyo, Japan) according to the manufacturer’s instructions. Rabbit polyclonal antiserum against formalin-fixed M. plutonius cells25 was diluted 8,192-fold in IHC diluent (primary Ab)  and used as the primary antibody to detect the antigen. The stained larval sections were observed under a light microscope .Surviving larvae were collected at days 2 and 4 pi (days 4 and 6 pg) for histopathological analysis. The larvae were then rinsed for several seconds with sterilized distilled water, fixed in 10% phosphate-buffered formalin, dehydrated in a series of ethanol, embedded in paraffin and sectioned using standard histological techniquespost-hoc test, and a value of P < 0.05 was set as the threshold for significance. All statistical analyses were performed using EZR 47, which is a graphical user interface for the R software48.The differences in the survival of tested larvae throughout the experiments were analyzed by the log-rank test and Bonferroni Supplementary Information.


The novel SARS-CoV-2 virus has led to a severe pandemic, starting from early 2020. Intensive care (ICU) management of the COVID-19 disease is difficult with high morbidity and mortality. Early nutritional support, especially with whey protein, seems to be crucial in this medical case. Thus, we aimed to assess the effects of an adequate nutritional protocol rich in whey protein on nutritional and inflammatory status, extubating time, and mortality of critically ill COVID-19 patients (CICP). Methods: A prospective single-center exploratory observational study was undertaken on 32 consecutive CICP admitted to the ICU of Santa Maria Hospital, Terni, Italy, and treated with whey protein-enriched formula. Patients’ demographics, nutritional status, indexes of inflammation, daily pre-albumin serum levels, duration of mechanical ventilation, and mortality were recorded. Results: Thirty-two patients were enrolled. Ninety-five percent of them showed a gradual reduction in C-reactive protein (CRP) values and increase in pre-albumin levels after the whey protein-enriched formula. Prealbumin levels were not correlated with a better nutritional status but with a shorter extubating time and better survival. Conclusions: An adequate administration of whey protein during COVID-19 patients’ ICU stays can provide fast achievement of protein targets, reducing the duration of mechanical ventilation, and improving inflammatory status and ICU survival. Further prospective and large-scale, controlled studies are needed to confirm these results. From January 2020, the novel corona virus (SARS-CoV-2) pneumonia COVID-19) spread around the world, causing a tremendous global health emergency and, then, a long-lasting pandemic. This novel viral agent causes, in a significant percentage of the infected patients, a serious bilateral pneumonia that resembles severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). The severity of the clinical condition has been reported to be milder than SARS with a mortality rate ranging from 4.3% to 11% [9 spread SARS-CoV-2 leads to a vast panel of different clinical manifestations. COVID-19 pneumonia is often associated with gastrointestinal disorders that compromise the patient’s ability to be adequately fed in the pre-Intensive Care Unit (ICU) stages [The data from GiViTi  show a direct correlation between inflammatory status, incidence of comorbidities, and mortality in these patients . The maiThe inflammatory status of COVID-19 patients is associated with an increased mortality ,4. MoreoOnce admitted to the ICU, nutritional risk could be monitored through a timely and longitudinal prealbumin and C-reactive protein (CRP) levels trends ,7. In moIn the ICU, the COVID-19 patients are mechanically ventilated for several days, turned into a prone position, then passed to non-invasive techniques, causing a sarcopenic and myopathy status . Thus, aThis prospective observational exploratory single-center study aims to evaluate the nutritional and anti-inflammatory effects of an outlined nutritional protocol based on enteral nutrition (EN) with a whey protein-based formula in COVID-19 patients admitted to the ICU. In detail, we aim to verify improvement of the nutritional risk and pre-albumin trends after this formula administration. Moreover, we aim to study the possible correlation between the pre-albumin levels and the duration of mechanical ventilation and mortality.In this single-center perspective exploratory study, we consecutively enrolled 32 COVID-19 adult patients admitted at the ICU of “Santa Maria Hospital“, Terni, Italy, between 9 March and 30 April 2020. We respected regional Ethical Committee rules for patients’ enrollment . Inclusion criteria were: age > 18 years, confirmed diagnosis of SARS-CoV-2 infection, and need of mechanical ventilation for at least 24 h. Patients were treated according to the present guidelines for COVID-19 .Starting of EN within 24 to 48 h from ICU admission, aiming to cover 50% of caloric needs;Ramping up to 80–100% of caloric needs after 48 h;Aiming a protein target ≥ 1.3 g/kg/day ,18;Aiming a caloric target of 20–25 kcal/kg/day;Using high-protein, 100% whey proteins, casein free, low in carbohydrate (CHO) content, and with a fat/CHO ratio of 50/50 EN formula, rich in Medium-Chain Triglycerides (MCT) and omega-3 fatty acids, with a complete vitamins, minerals, and trace elements profile.According to a standardized nutritional protocol for COVID-19 patients, each patient received EN applying the following nutritional scheme ,18:StarCalories:11–14 kcal/kg current body weight/day, for BMI of 30–50;22–25 kcal/kg IBW, for BMI > 50;65–70% of the measured calories.Protein intake:2 g/kg IBW for BMI 30–50;2.5 g/kg IBW for BMI > 50.EN was also administered to the patients during prone positioning, maintaining at least 10% of bed anti-Trendelenburg tilt, and to the patients treated with NIV and in the conditions of controlled hypoxemia and permissive hypercapnia. EN was started at 20 mL/h infusion rate and increased by 10 to 20 mL/h every 24 h, based on the gastric tolerability. The feeding rate was reduced in case of >500 mL/24 h, adding pro-kinetics (i.v. metoclopramide 10 mg t.i.d.), and evaluating the correct positioning of the nasogastric tube and the needs of supplementary parenteral nutrition. In case of 1% propofol infusion higher than 20 mL/h, the calories and amount of lipids provided with this drug were taken into account in the daily energy balance. Concerning the obese patients, nutritional targets were calculated considering Body Mass Index (BMI) and Ideal Body Weight (IBW) as follows:The needs of trace elements and vitamins supplementation were evaluated in previously malnourished patients, or in case of partial EN with or without complementary parenteral nutrition  ,18.® Metabolic Monitor . Patients admitted to ICU were temporarily excluded from indirect calorimetry (IC) assessment under the following conditions: fraction of inspired oxygen (FiO2) > 70%, hemodynamic instability, positive end-expiratory pressure (PEEP) > 16 mmHg on active veno-venous extracorporeal membrane oxygenation (VV ECMO), or per ICU attending clinical judgment. IC data were selected from 10 to 30 min intervals, which met steady-state conditions defined by a variance of volume of oxygen (VO2) and volume of carbon dioxide (VCO2) by <10% as per published validation data for the Q-NRG device [The mREE data were obtained using the Q-NRG+G device . The patAll the clinical and laboratory data were prospectively collected from the patient’s medical file. General and demographic variables on the day of ICU admission were recorded and the SAPS II score was calculated in order to obtain the information on the severity of critical illness . All theStatistical analyses were performed using SPSS Software 21 . Quantitative variables distribution was tested with Kolmogorov–Smirnov normality test. All data are presented as mean ± standard deviation (SD) or median  according to the normal or not normal distribution. Parametric (Student’s T-Test) and non-parametric tests (Mann–Whitney U test) were applied to describe the differences between groups for the variables of interest as appropriate. Kaplan–Meier survival curves for the comparison of the Hazard Ratio between groups were built for the survival analysis. The alpha level of significance was set at 0.05 [From 9 March to 30 April 2020, 32 ICU SARS-CoV-2 infected patients meeting the inclusion criteria were consecutively enrolled. The study population had a mean age of 68 ± 12.5 years, with a mean SAPS II score of 57 ± 12 points at the admission to the ICU. Considering inflammatory/infection indices at day 1, median procalcitonin was 0.37 (0.19–1.29) ng/mL, median CRP was 19 (5.6–31) mg/L; white blood cells count 8070 6263–11,000). ICU mortality was 31.3% 10 patients). Non-survivors were significantly older and severely ill as shown in  patients,000. ICUBecause of high formula tolerability, it was possible to administer all the patients with the following average intake: mean daily caloric intake: 20 to 30 kcal/kg/day; mean daily protein intake: 1.3 g protein/kg/day.p = NS) (p < 0.05).The first section of the analysis was focused on pre-albumin values during the ICU in the study population. Median serum pre-albumin level at the ICU admission was 9 (range 6–14) mg/dL. The 59.4% of the patients (19 out of 32) had a pre-albumin level below 10 mg/dL threshold at the ICU admission, as a possible sign of pre-existent state of malnutrition. This finding was only partially in agreement with indirect calorimetry assessment that showed a mild tendency toward hypermetabolism . There was no statistical difference between survivors and non-survivors (p = NS) . This vap < 0.05) (Twelve patients (63.2%) were able to reach and maintain the pre-albumin threshold during the ICU stay, while one patient reached that at a certain point of the ICU stay but did not maintain that. Six patients (31.6%) did never reach the threshold despite the application of the nutritional protocol. In all the 32 analyzed patients, there was a trend toward increase in pre-albumin levels during the ICU stay. This trend was not shown in three patients belonging to the group of non-survivors  .p = NS)  . Other ip = NS) (p < 0.05). This was not observed in non-survivor group (p = NS).In order to investigate the percentage of the patients reaching different thresholds of serum pre-albumin concentration during their ICU stay, the threshold was defined as reached: if at least 75% of subsequent measurements were not below the threshold itself, then it was assumed that the patients had maintained the considered threshold. In detail, 81.3%, 62.5%, and 53.1% of the study population reached the threshold of 10 mg/dL, 15 mg/dL, and 20 mg/dL, respectively (p = NS) . InteresThe p < 0.00001) . Finally, looking at the survival analysis following Kaplan–Meier, the patients who were able to reach and maintain the serum pre-albumin level threshold > 10 mg/dL showed a greater ICU survival vs. other patients  .p < 0.0001).All surviving patients reached a target of protein intake of 1.3 g/kg/day. The median achievement time was 3 ± 0.3 days (range 2–6). Only one patient in the “non-survivors” group could reach the protein intake target; the target time was of 3 ± 0.4 days. The most severely ill patients (identified by a higher SAPS II) appeared to require a longer time to reach the protein target (PT) . Interesp < 0.05)  .The present prospective observational study has primarily been aimed to investigate the impact of whey protein-based EN formula on nutritional, inflammatory markers, extubation time, and survival rate of SARS-CoV-2 critically ill patients. Pre-albumin values significantly increased, and this was followed by CRP reduction and a better survival rate, rather than nutritional status. The application of a feeding protocol based on whey protein-based formula seems to offer a survival advantage to these patients. In detail, the majority of the patients were able to reach the protein intake target and the time to reach this target showed an association with the extubation time. Moreover, this EN regimen allowed to reach normal serum pre-albumin levels in a vast majority of the study population and this was associated with lower ICU mortality and a reduction in the duration of mechanical ventilation.However, IC data did not agree with pre-albumin levels during the ICU stay. Indeed, pre-albumin is a controversial marker of malnutrition and nutritional risks both in COVID-19 and non-COVID-19 patients . MalnutrPre-albumin is a tetrameric protein secreted mainly by the liver and choroidal plexus and accounts for 15% albumin serum level . Due to pre-albumin short half-life (2 days) vs. albumin (14–20 days), small volume of distribution and uncomplicated measurement, it could be a good candidate for monitoring rapid changes in metabolic status ,31. SeveIn this study population, a relevant number of the patients was admitted to the ICU with low serum pre-albumin levels. This could be interpreted as a sign of diffuse malnutrition status in these patients even before the ICU admission. However, we must recognize that COVID-19 patients arrive at the intensivist attention after days of illness, treated both at home and in general wards. More in detail, COVID-19 is often associated with fever, lack of appetite, and various gastrointestinal disturbances such as nausea, vomiting, and diarrhea. These conditions can bias and influence the values of pre-albumin at the moment of ICU admission. Other biasing factors are the large use of CPAP masks and NIV that can substantially contribute to malnutrition and muscle waste before the ICU admission. Furthermore, severe protein degradation may lead to unintended and extensive muscle wasting (up to 15–25% of the muscle mass), recently quantified during the first ten days after ICU admission [p = 0.033). Scientific society guidelines and recommendations are in favor of this practice. More in detail, a sufficient (high-dose) protein load should be provided to critically ill patients with a protein requirement expected to be in the range of 1.2–2.0 g/kg actual body weight per day. These aspects should be taken into great account when approaching COVID-19 patients and their nutritional status and risk of rapid malnutrition development. Moreover, high-protein formulas and protein derivatives can have direct anti-viral and anti-inflammatory effects in COVID-19 [In this study, we observed that a whey protein-enriched nutritional scheme can reverse the vicious circle of protein depletion related to both ICU admission and COVID-19 clinical course. In a prospective database of 843 ICU patients undergoing mechanical ventilation , a signiCOVID-19 . The vasp < 0.0001), increased ICU stay , and increased mortality  [In literature, there are different findings on the efficacy of a high-protein formula in reaching such results on mortality and morbidity. Alberda et al. carried out a multicenter study on more than 2770 ICU mechanically ventilated patients . Interes = 0.04) . We have = 0.04) .In this study, the pre-albumin trend was significantly correlated to an increased nutrition and mortality risk . The visThis study has several limitations to be acknowledged. The first, and main limitation, the single-center exploratory design does not allow the authors to draw definitive conclusions regarding the real impact of this EN whey protein-enriched protocol in COVID-19 patients. An interventional control group is missing, perhaps one using a different EN formula. This has been conditioned by the need to provide the best EN protocol to COVID-19 patients in this pandemic. Second, the small sample size and lack of a control group  do not allow the authors to establish a direct causality connection between the investigated variables and outcome. Third, pre-albumin is not a reliable nutritional status biomarker, and findings from the present study confirm this evidence. On the other hand, pre-albumin has been confirmed as a “hybrid” biomarker for mortality and morbidity of ICU patients. Findings from this study need to be confirmed by larger multicentric controlled trials.In conclusion, the application of a nutritional protocol with a highly tolerated enteral formula based on whey protein, suggests a positive effect on patients’ outcome and a positive effect on the duration of mechanical ventilation in SARS-CoV-2-infected critically ill patients. Pre-albumin seems to be useful in survival rate prediction in ICU patients.The anti-inflammatory properties of whey proteins could have an important adjunctive role in the treatment of high inflammation-generating infections such as the new SARS-CoV-2 virus. These preliminary results will ensure further larger prospective controlled investigations in this field.


Background and aimsNon-invasive assessment methods to assess liver fibrosis are important tools where FibroScan or liver biopsy is not accessible. The aim of this study is to assess the efficacy and performance of the fibrosis index based on four factors (FIB-4) and aspartate transaminase-to-platelet ratio index (APRI) to evaluate liver fibrosis against FibroScan for the stages of liver fibrosis in patients of chronic liver disease due to chronic hepatitis B (CHB).MethodsThis was a cross-sectional study conducted in a tertiary care center in Uttar Pradesh, India, and the patients were enrolled between 2017 and 2020. During the study period, 520 patients with a confirmed diagnosis of chronic hepatitis B virus (HBV) infection were selected. Laboratory blood testing and FibroScan were performed in all patients with CHB. APRI and FIB-4 were calculated using a standard formula involving laboratory parameters.ResultThe performance of FIB-4 scores are nearly similar to APRI, with area under the curve (AUC) 0.753, (95% CI) (0.711-0.795) (p<0.0001) for ≥F2 fibrosis (significant fibrosis) and even better 0.851 (0.815-0.887) (p<0.0001) for the F4 fibrosis (cirrhosis) group. Both the tests are proven good to diagnose fibrosis but FIB-4 has more area under the receiver operating characteristic (AUROC) than APRI in each set, thus FIB-4 is considered better than APRI.ConclusionsAPRI and FIB-4 scores showed good performance in detecting patients without liver fibrosis as compared with FibroScan. Based on this study, FibroScan can be avoided in patients examined for the diagnosis of mild fibrosis and cirrhosis in the source constrained area. Hepatitis B virus (HBV) is an infectious agent that infects the human liver cells and causes liver inflammation, which leads to chronic infection and severe problems such as liver cirrhosis (LC) or hepatocellular carcinoma (HCC) more frequently than the other types of hepatitis viruses. The World Health Organization estimated that the number of people exposed to the hepatitis B virus to be approximately 2 billion ; 240 milProgressive hepatic fibrosis with the development of cirrhosis is a characteristic of almost all chronic liver diseases. In patients with CHB, the accurate stage of hepatic fibrosis is the most important predictor of disease progression and indicates the need for initiating antiviral therapy. For many years, liver biopsy has been considered the gold standard for the staging of fibrosis . HoweverFibroScan has been introduced as a new, non-invasive method for the diagnosis of liver fibrosis. FibroScan is based on the principle of transient elastography (TE), that is, the propagation velocity of a wave through a homogenous tissue is proportional to its elasticity, which is correlated with the amount of fibrosis in the liver . FibroScMany studies report that FibroScan could predict liver fibrosis accurately in patients with chronic hepatitis C (CHC) -13. In rVarious methods including serum markers, such as AST platelet ratio index (APRI), FIB-4, and TE (FibroScan) ,16, haveThere are many advantages of non-invasive tests for detecting fibrosis, such as easy-to-perform and widespread availability, almost free from complications, can be done in the outpatient setting, does not require specialized training, homogeneity because of automated measurements of their component variables, tools for automated computation of score available (phone apps), and easy to repeat at frequent intervals. Because of the risks, contraindications, and observer dependence associated with liver biopsy, non-invasive methods should be better to evaluate liver fibrosis. Few non-invasive methods, such as the fibrosis index based on four factors (FIB-4), sonographic transient elastography (FibroScan), and aspartate aminotransferase (AST), have been intensively developed and have improved evaluation of the LF (liver fibrosis) stages.The aim of this study was to evaluate the effectiveness of FIB-4 and APRI to differentiate the stages of liver fibrosis against FibroScan-based staging of liver stiffness in patients with chronic hepatitis B (CHB) infection. We also tried to evaluate whether any non-invasive methods are superior among both of them or not.Study period, population, and designWe performed a cross-sectional study from 01 January 2017 to 30 January 2020 in the department of medical gastroenterology, King George Medical University, Lucknow, Uttar Pradesh, India. During the study period, 520 patients with a confirmed diagnosis of chronic HBV infection were enrolled. This study was conducted following recommendations of the ethics research committee (registration no. ECR/262/Inst/UP/2013/RR-16) and ethical clearance  was obtained from our institution. Written consent was also obtained from all the participants for conducting the present study.All patients were asked about their exposure to risk factors , presence of ascites, jaundice, hematemesis, melena, pedal edema, easy bruisability, bleeding gums, or recent use of any alternative medicine or alcohol. This study included both acute HBV and chronic HBV-infected patients with and without cirrhosis, as well as patients who were on antiviral therapy and follow-up patients who were not on antiviral therapy.Patient’s history, physical examination, hematological and biochemical investigations like hemogram, liver function tests, serum protein and albumin tests, ultrasonography (USG), and TE were done in all the patients. Patients with the presence of other causes of liver disease, HCC, prior interferon therapy, human immunodeficiency virus (HIV), and co-infected with HBV, and liver transplantation were excluded from the study.Laboratory methodsHematological and biochemical parameters were determined using commercially available assays. All patients' samples were tested for HBsAg by using a commercial enzyme-linked immunoassay (ELISA) kit . All HBV-positive patients were further investigated for quantitative HBV DNA by reverse transcription-polymerase chain reaction (RT-PCR) . Further, the included patients were investigated for HBeAg and HBeAb.Liver stiffness measurementsTransient elastography (FibroScan) was performed on all of the patients in this study  technology). According to the manufacturer's instructions, a test must have 10 correct FibroScan readings to be considered accurate.Stages of liver fibrosis were classified as follows based on FibroScan scores: F0-F1 (7 kPa), F2 (7-8.99 kPa), F3 (9-12.49 kPa), and F4 or cirrhosis (12.50 kPa). For each patient, FIB-4 and APRI scores were calculated based on laboratory results and the values were rounded to two decimal places. To predict patients with fibrosis (METAVIR≥F2), a lower and upper cutoff value of 1.45 and 3.25 for FIB-4 and 0.5 and 1.5, respectively, for the APRI were used based on the available data from the WHO guideline. However, APRI values of 1.0 and 2.0 are the lower and upper cutoff for detecting patients with cirrhosis, respectively, while no cutoff value for FIB-4 has been found to be satisfactory for detecting cirrhosis (METAVIR F4) .Non-invasive scores (APRI and FIB-4) were calculated using laboratory data based on the following formulas:APRI = [{AST (IU/L) / Upper normal limit of AST (IU/L)}/Platelet count (109/L)]*100 FIB-4 score = Age (years) X AST (IU/L) / Platelet count (109/L) * ALT (IU/ L)1/2 Statistical analysisIBM’s SPSS version 24.0 software was utilized to conduct statistical analysis and to draft the data. For descriptive analysis, median and interquartile range (IQR) were obtained, non-parametric continuous variables, and percentages and numbers were obtained for categorical variables. The diagnostic performance of APRI and FIB-4 scores was measured by the area under the receiver operating characteristic (ROC) curve. The balance between sensitivity (Se), specificity (Sp) for a particular value of the test to rule out, or rule in the patients of interest was obtained from the coordinates of the curve. Positive predictive values (PPV) and negative predictive values (NPV) were also obtained for the cutoff value of the test. Statistical significance was defined as p<0.01.Baseline dataOut of the total 520 patients enrolled in this study, around two-thirds (73.8%) of patients were male and one-third (26.2%) were female; however, this difference is statistically insignificant (p=0.82). The mean age of the study population was 36.36±14.39 years. The baseline biochemical characteristics of the study population are presented in Table For the study purposes, we made two sets of population groups. In the first set, we divided the study population into patients with normal liver  and patients with any kind of liver fibrosis or cirrhosis (i.e fibroscan ≥7 kPa); the second set of non-cirrhotic patients  were included in group 1 and cirrhotic patients included in group 2 .In set one, out of 520 patients, 248 (47.7%) had normal liver and 264 (52.3%) patients had fibrosis or cirrhosis while in set two, out of 520 patients, 390 (75%) patients were having non-cirrhotic liver and 130 (25%) had cirrhotic liver.Diagnostic performance of APRI and FIB-4 for significant fibrosis and cirrhosisHigh AUROC values for APRI and FIB-4 indicated the very good performance of these tests in recognizing significant fibrosis and cirrhosis. AUROC of APRI and FIB-4 to discover significant fibrosis ≥F2) were 0.756  and 0.753 (95% CI 0.711-0.795), respectively. For the diagnosis of cirrhosis AUROC of APRI and FIB-4 were 0.818 (95% CI 0.776-0.861) and 0.815 (95% CI 0.815-0.887), respectively. The performance of APRI and FIB-4 on ROC are plotted in Table  were 0.7The FIB-4 score are nearly similar to APRI, with AUC mean (95% CI) 0.753 0.711-0.795) (p<0.0001) for significant fibrosis and even better, i.e. 0.851 (0.815-0.887) (p<0.0001) for the cirrhosis group  is a major public health issue that causes substantial morbidity and mortality. The progression and stage of liver fibrosis determine the prognosis and treatment of the disease. For clinical decision-making and follow-up, accurate quantification of liver fibrosis is of paramount importance. Only a liver histological examination can accurately confirm the existence of concomitant liver fibrosis, necroinflammatory activity, and steatosis. Liver biopsy is the gold standard test for evaluating liver fibrosis stages and due to the invasive nature of the procedure, the small size of the specimen, sampling risks, and inter-observer variability in the histopathological examination can restrict the routine use of liver biopsy . Due to Due to the non-availability and high cost of transient elastography (FibroScan) in peripheral health centers made clinicians think of some other non-invasive reliable methods to determine liver fibrosis. APRI and FIB-4 are well-studied, non-invasive methods that can access liver fibrosis and cirrhosis .In present study APRI identified significant fibrosis p<0.0001) with a related AUC mean (95% CI) 0.756 (0.714-0.797)  that assessed APRI and found the area under the summary receiver operating characteristic (SROC) curve to be 0.77. In the same study, a summary receiver of operating characteristic curve of meta-analysis of 11 studies  that assessed APRI for cirrhosis and found the area under the SROC curve to be 0.75 while our study finds AUROC for the same was 0.818, which is far better . We alsoIn this study, the ROC of FIB-4 was higher than that of APRI  for diagnosing cirrhosis, which indicated FIB-4 might be more reliable than APRI as an indicator of cirrhosis in HBsAg-positive CHB patients with ALT≤5 ULN. Thus this study confirms the better performance of FIB-4 to predict cirrhosis with an AUROC of 0.851 in HBsAg-positive CHB patients.For the detection of significant fibrosis, as described earlier, AUROC was similar for APRI and FIB-4  in this study but only sensitivity for lower and upper cut-offs of APRI are found to be better as compared to WHO for the same (87 vs 82% and 54.7 vs 39%) .We also compared our study population between cirrhotic and non-cirrhotic at a lower cut-off of 1.0 and an upper cut-off of 2.0 for APRI as described by the WHO guideline against the METAVIR scoring system, and we found that sensitivity for lower cut-off (80.8 vs 77%), i.e. higher than WHO results, specificity for lower cut-off (76.9 vs 78%) is almost similar, while sensitivity and specificity for upper cut-off (51.5 vs 48% and 90.5 vs 94), i.e. almost near to the WHO guideline (Table Some studies show different pathogenesis and different patterns of fibrosis according to different causes of chronic liver diseases and also justify the need for different cut-points of systems for the assessment of fibrosis from different causes -29.APRI and FIB-4 were used primarily in resource-limited areas to predict liver fibrosis and cirrhosis . In the Based on evidence from the systematic review, the WHO guidelines recommended that FibroScan and APRI were the most useful tests for the assessment of cirrhosis in resource-limited settings . Our resThere may be a bias in the retrospective design of this study that might have caused selective bias resulting in underestimated sensitivity and overestimated specificity of APRI and FIB-4. Therefore, a larger sample with multicenter studies will be necessary to validate the new cut-offs of APRI and FIB-4.Since liver cirrhosis is the driving factor in CHB infection to determine the treatment regimen, duration, and follow-up strategy, the fibrosis test should be able to differentiate the maximum number of cirrhotic and mild fibrosis from normal or early stages of fibrosis. APRI and FIB-4 scores also showed good performance in detecting the patients without liver fibrosis compared with FibroScan. In conclusion, FIB-4 should be considered as the preferred non-invasive fibrosis test, and FibroScan should be considered when FIB-4 is unavailable. Based on this study, a liver biopsy could be avoided in patients examined for the diagnosis of significant fibrosis and cirrhosis.


Cordyceps militaris (CM) proteome using machine-learning-based prediction servers for anticancer prediction, i.e., AntiCP, iACP, and MLACP. The selected CM-anticancer peptide candidates could be an alternative treatment or co-treatment agent for colorectal cancer, reducing the use of chemotherapeutic drugs. To ensure the anticancer properties, an in vitro assay was performed with “CM-biomimetic peptides” on the non-metastatic colon cancer cell line (HT-29). According to the 3--2,5-diphenyltetrazolium bromide (MTT) assay results from peptide candidate treatments at 0–400 µM, the IC50 doses of the CM-biomimetic peptide with no toxic and cancer-cell-penetrating ability, original C. militaris biomimetic peptide (C-ori), against the HT-29 cell line were 114.9 µM at 72 hours. The effects of C-ori compared to the doxorubicin, a conventional chemotherapeutic drug for colon cancer treatment, and the combination effects of both the CM-anticancer peptide and doxorubicin were observed. The results showed that C-ori increased the overall efficiency in the combination treatment with doxorubicin. According to the acridine orange/propidium iodine (AO/PI) staining assay, C-ori can induce apoptosis in HT-29 cells significantly, confirmed by chromatin condensation, membrane blebbing, apoptotic bodies, and late apoptosis which were observed under a fluorescence microscope.Colorectal cancer is one of the leading causes of cancer-related death in Thailand and many other countries. The standard practice for curing this cancer is surgery with an adjuvant chemotherapy treatment. However, the unfavorable side effects of chemotherapeutic drugs are undeniable. Recently, protein hydrolysates and anticancer peptides have become popular alternative options for colon cancer treatment. Therefore, we aimed to screen and select the anticancer peptide candidates from the in silico pepsin hydrolysate of a  Colorectal cancer is the third most common cancer globally . Even thCordyceps militaris (CM) has been widely used as a nutrient supplement in eastern Asia, and is extensively applied in modern medical therapeutic methods [ methods . CM pept methods . BecauseCordyceps militaris peptide dataset. AntiCP utilized the amino acid composition and binary features, with SVM algorithm support, to classify the ACPs. The database of AntiCP was based on a dataset of 225 antimicrobial peptides (AMPs) with anticancer properties and random peptides from SwissProt  [In this study, we proposed the use of support vector machine (SVM)-based predictors and the ensemble approach to identify the potential ACP candidates from the ay 2021) . MLACP day 2021) . These pEven though ACPs have many advantages, they also contain many shortcomings which diminish the peptides’ efficiency . The recTo evaluate the anticancer activities of the anticancer candidate peptides, in vitro methods were utilized to confirm viability inhibition against colon cancer cell lines. The fluorescence measurement was also used to investigate the activity of peptides against the cells and identify the modes of action. Thus, our bioinformatic analysis provided ACP identification from nearly twenty thousand peptides, and the in vitro result showed the effects of peptides on cell lines.The peptides were predicted to be anti-cancer peptides (ACPs) if: the cut off criteria at the support vector machine (SVM) score was over 1.00 in AntiCP; the probability was over 0.5 in iACP; and the scores of both random forest (RF) and SVM probabilities were over 0.5. As a result, AntiCP predicted 20,525 ACPs; iACP predicted 3,739 ACPs; MLACP predicted 1,181 ACPs; and three prediction servers predicted 316 ACPs in common from 21,148 unique CM peptides. In terms of the peptides’ properties , the majhttps://webs.iiitd.edu.in/raghava/toxinpred/protein.php, accessed on 12 May 2021) and MLCPP  were used to predict the modes of action of the putative ACPs. ToxinPred predicted the toxicity of peptides against cancerous cells, while MLCPP predicted the cell penetration ability of peptides against cancer. Both ToxinPred and MLCPP predicted 14 peptides to be toxic ACPs; 242 peptides to be cell-penetrating ACPs; 5 peptides to have both modes of action; and 55 peptides to have no mode of action. We were interested to investigate the difference in the effects between peptides with no modes of action and with both modes of action.ToxinPred , and SVM-ACP). As a result, TTMICLTCAR was the candidate for the original toxic and cancer cell-penetrating peptide named “CTP-ori”. CTP-ori had ten amino acids consisting of three polar uncharged threonines, two cysteines, and one positive charged arginine. CTP-ori was a hydrophilic, amphiphilic, and cationic peptide. To increase the prediction score, CTP-ori was re-designed by substituting certain amino acid residues using the peptide design option on AntiCP. CTP-ori was modified into a CM-biomimetic toxic and cancer cell penetration peptide, or “CTP-rds”, by replacing methionine and alanine with glycine and cysteine, respectively (TTGICLTCCR) .From all 55 peptides with no special mode of actions against cancer cells, VTFVLIAAK was the candidate for the original CM-biomimetic peptide named “C-ori”. C-ori had nine amino acids with double alanine residues, one polar uncharged threonine, one aromatic residue , and a positive charged lysine (VTFVLIAAK). The physiochemical properties of C-ori were hydrophobic, amphipathic, and cationic. The modified CM-biomimetic peptide, C-rds, was the result of AntiCP’s modification by having the first valine and isoleucine replaced by phenylalanine and leucine, respectively (FTFVLLAAK) .In accordance with the bioinformatics prediction, we decided to synthesize four putative ACPs .All selected peptides’ molecular structures were predicted using PEP-FOLD3.5. The C-ori was random coiled c, wherea50 of C-ori was 114.9 ± 1.5 µM  . For exaAs can be seen in the pipeline illustrated in http://crdd.osdd.net/raghava/anticp, accessed on 12 May 2021), iACP , and MLACP . Venny 2.1.0.  was used to generate the Venn diagram to visualize the unique, dual, and multifunctional peptide candidates from each prediction online bioinformatic program. ToxinPred  and MLCPP  were used to predict whether the peptides were cytotoxic, or if they were cell-membrane-penetrating anti-cancer peptides or not. The selected putative ACPs were redesigned using AntiCP, utilizing the peptide design function. Lastly, PEP-FOLD3.0  was used to simulate the feasible molecular structure of the peptides.A total of 21,148 CM peptide sequences were obtained from the predicted cut site of the contig1 CM proteome , and the in silico pepsin digestion was performed by our Python scripts. There were 3 groups of peptide length distributions, i.e., 5–20 amino acid residues , 21–35 amino acid residues , and 36–50 amino acid residues  . The pep2.The non-metastasis colorectal cancer cell line HT-29 and the human dermal fibroblast cell were kindly provided as a gift from Dr. Mattaka Khongkaw, senior researcher from National Nanotechnology Center (Nanotec) Thailand. HT-29 and the human dermal fibroblast cell were cultured and passaged in Dulbecco’s Modified Eagle Medium , and supplemented with a 10% heat-inactivated Fetal Bovine Serum  at 37 °C under 5% CO5 cells/well (100 µL/well) for 24 h, and treated with indicated concentrations of C-ori and C-rds (0 to 400 µM) for 24 h, 48 h, and 72 h, while the cells were treated with CTP-ori and CTP-rds at a different range of concentrations (0 to 100 µM) due to the limited quantity of the peptides. After it was due, MTT was added to each well with the same concentration of 1 mg/mL (10 µL/well) and was further incubated for 4 h. The media were removed and 100 µL of DMSO was added to dissolve the purple crystal. Absorbance was measured at 570 nm using the absorbance microplate reader, PowerWave HT . The cell viability was calculated using the following formula:The cell viability was evaluated by a conventional 3--2,5-diphenyltetrazolium bromide (MTT) assay. The selected CM-biomimetic peptides were synthesized by Cellmano Biotech Limited . The peptides were dissolved by dimethyl sulfoxide  until the final concentration was 100 mM. Cells were preincubated in 96-well plates at 2 × 1050) was estimated by fitting the data with the non-linear regression equation.The half-maximal inhibitory concentration  if not stated otherwise. The parameters were analyzed with a one-way analysis of variance (one-way ANOVA) followed by Dunnett’s multiple comparisons test. Graphpad Prism Version 9.0  was utilized to conduct all statistical analysis. The values of The chemotherapy drug, doxorubicin hydrochloride , was used to compare the effects with the selected peptide. The condition of the setting experiment was similar to the cell viability determination method. The cells were treated with different concentrations of doxorubicin (0 to 0.25 µM) in a two-fold manner to determine the cytotoxicity of doxorubicin. The combination of C-ori (0 to 400 µM) and doxorubicin (0 to 0.25µM) was treated on cells in a two-fold manner to observe the effect of the combination. The cell viability was evaluated using an MTT assay following the above-mentioned procedures in 4 cells/well (400µL/well), and was allowed to adhere at 37 °C in a 95% relative humidified incubator containing 5% CO2 for 24 h. The HT-29 was exposed to different concentrations of various drugs  for 72 h. The adherent HT-29 cells were trypsinised and centrifuged  at 1000 rpm at 4 °C for 10 minutes. The pallet was washed using phosphate-buffered saline (PBS), and re-centrifuged three times with 1000 µL of PBS; afterwards, the PBS and waste were removed. Then, 5 µL of PBS and 5 µL of both 1 mg/mL acridine orange and 10 mg/mL propidium iodine were mixed with the pallet. The aliquots were visualized under a fluorescence microscope .An acridine orange/propidium iodine (AO/PI) double staining assay was employed to determine the effects of the peptides on the HT-29 cancer cell death. HT-29 was seeded in an 8-well plate at 5 × 10Cordyceps militaris which exhibited the apoptogenic effect against non-metastasis colorectal cell lines (HT-29). C-ori can be modified to increase the efficacy of peptides and can be developed to be used as a co-treatment with the conventional chemotherapy in clinical use. Even though the observed anticancer activity of the C-ori was rather weak and not quite suitable for further clinical trials, the anticancer activity of the designed peptide could be improved with drug delivery systems to be more specific to cancer cells with an improved uptake rate. Moreover, the non-effective biomimetic ACP candidates in our work might still have other potential uses as anti-cancer cells, e.g., cell cycle arrest or senescence induction, which are other modes of action that need further investigation. Furthermore, the mechanisms of C-ori in inhibiting other types of various cancer cell lines, as well as the in vivo method conducted on sample animals, have to be further studied.In conclusion, C-ori was the original pepsinized anticancer peptide from


Despite different genetic background, Noonan syndrome (NS) shares similar phenotype features to Turner syndrome (TS) such as short stature, webbed neck and congenital heart defects. TS is an entity with decreased growth hormone (GH) responsiveness. Whether this is found in NS is debated.® 33 or 66 μg/kg/day, and TS-girls were consecutively treated with Genotropin® 33 or 66 μg/kg/day.Data were retrieved from combined intervention studies including 25 children diagnosed with NS, 40 diagnosed with TS, and 45 control children . NS-children and TS-girls were rhGH treated after investigation of the GH/IGFI-axis. GH was measured with poly- and monoclonal antibodies; 24hGH-profile pattern analysed by PULSAR. The NS-children were randomly assigned to Norditropinmax24h-profile (Mean ± SD) was higher in both groups  as was GH-baseline . Pre-treatment IGFISDS was 2.2 lower in NS-children (-1.7 ± 1.3) compared to TS-girls . GHmax, IGFI/IGFBP3-ratioSDS, and chronological age at start of GH accounted for 59% of the variance in first-year growth response in NS.Higher PULSAR-estimates of 24h-profiles were found in both NS-children and TS-girls compared to controls: Polyclonal GHBoth prepubertal NS-children and TS-girls had a high GH secretion, but low IGFI/IGFBP3 levels only in NS-children. Both groups presented a broad individual response. NS-children showed higher response in IGFI and growth, pointing to higher responsiveness to GH treatment than TS-girls. Individuals with syndromes, like Turner syndrome (TS), have been reported to have reduced growth hormone (GH) responsiveness - partially overcome by higher rhGH dosage . MoreoveDue to the phenotypical resemblance, NS has previously been called “male Turner syndrome (TS)” , despitemax levels in response to stimulatory test. Still, all of these studies found lower level of IGFI.One of the most predominant features of children with NS and TS is short stature affecting more than 80% . PossiblSo far, only two studies have displayed detailed analyses pattern of the spontaneous GH secretion and its correlation with growth in NS patients treated with rhGH. Both Ahmed et al.  with a pOur overall aim was to investigate GH/IGF status, response in growth and IGFI, and responsiveness to rhGH treatment in children with NS compared to TS. In this paper, we present unique and detailed data on the 24hGH profile in 25 prepubertal children with NS, and we compare these data to that of girls with TS and to healthy normally growing children as a control group. Both growth response and response for IGFI and IGFBP3 during the first year of GH treatment were measured and compared. Our hypothesis was that children with NS have disturbed GH secretion but normal GH responsiveness, giving greater response on a similar dose of rhGH treatment compared to girls with TS.Noonan children: This study group comprised 25 prepubertal patients with NS  all included in the formal trial GHN00-1658 and were followed prospectively from pre-treatment to adult height. The entry criteria were postnatal growth restriction leading to short stature, webbed neck, ptosis, facial dysmorphism, low posterior hairline, congenital heart defects but no cardiomyopathy, normal karyotype, hematologic anomalies, normal liver and kidney function and delayed bone age  ( 87-052) –22. All Healthy children: Data from a reference group of 45 healthy children  with normal height  with non-thrombogenic catheter  were used to collect blood samples as previously described .The IGFI and IGFBP3 serum concentrations were measured before starting treatment (n=25 for NS and n=30 for TS), at an average of 10 days (range between 7-21 days) in 15 NS and in 24 TS, and also after one year of treatment in 23 NS and in 24 TS girls. These blood samples were collected after noon, and approximately 24h after the latest GH injection.®/Novo Nordisk in all NS and Somatonorm® in 4 or Genotropin®/Pfizer in 36 TS) was given in a dose of 33 µg/kg/day in 9 patients with NS  and in 32 patients with TS, and 66 µg/kg/day in 16 patients with NS , and in 8 patients with TS. The dose was randomized in 16 NS, whereas the dose in TS was chosen consecutively with a lower dosage of 33 µg/kg/day in the first 32 patients and later with a higher dosage of 66 µg/kg/day in 8 patients. The doses were adjusted according to changes in body weight every 3 months. Details on NS group are reported elsewhere  80/505 . During 1991 and 1992 the laboratories in Sweden switched from WHO IRP 66/217 to WHO IRP 80/505; a conversion factor of 1.55 had to be used to compare the results on GH levels . All girThe analysis of the 24hGH profiles was performed as previously reported for pulse detection and peak analysis with the PULSAR program, with settings for GH-profiles , 28. PULThe serum concentrations of IGFI were measured at start of GH treatment, at an average of 10 days (range 7-21 days), and at 12 months after start of GH treatment. An IGF-binding protein (IGFBP)–blocked radioimmunoassay (RIA) without extraction and in the presence of an approximately 250-fold excess of IGFII  was used . Serum IThe Ethic Committee of the Faculty of Medicine at the University of Gothenburg approved the Noonan study GHN00-1658 (Dnr 208-05), the healthy children study (Dnr 129-85), and for the Turner studies TRN 88-072 and TRN 87-052 the Gothenburg part  of the multicentre studies approved by the Ethics Committee of Lund University. Informed consent was obtained from the parents and from patients if they were old enough.All data are presented as mean ± SD, if not otherwise stated. Mann-Whitney U-test for continuous variables was used for comparison between two groups. Analyses were adjusted for pretreatment status and rhGH dose in the multiple regression analyses with a normally transformed dependent variable, Blom’s method. Spearman correlation coefficients and corresponding p-values were obtained when describing relation between two continuous variables.SDS with SDS for IGFI, IGFBP3 and the ratio, multiple regression analyses were done with interaction term of predictor and group variable (NS or TS) added to the model. These analyses were also adjusted for rhGH dose and the p-value for the interaction term tested if the slopes were significantly different between the two groups.To test the difference of slopes, for prediction of delta heightSDS first year of GH-treatment within NS and TS by initially including all significant predictors from univariate analyses. All tests were two-tailed and conducted at 5% significance level.Finally, multiple stepwise regression models were performed for prediction of delta heightGH profile: The GH mean, GHmax, AUCt, AUCb, and the GH-baseline were significantly higher in both NS and TS compared to healthy children. The average number of GH peaks during a 24h period was higher only for NS patients compared with TS and healthy children, respectively. In the TS group, the peak width was statistically greater than in the healthy group. See vs. 4.6±2.3 mU/L; p<0.05), GHmax , GH-baseline , number of peaks , AUCb , and AUCt . Girls with NS did not show any significant difference in the 24hGH profile when compared to healthy girls. See When compared to healthy boys, boys with NS displayed significantly greater mean GH . The polyclonal/monoclonal GH ratio had extremely high levels of non-22kDa-isoforms in three females and one male. For the entire NS-group, this ratio was for GHmax 1.13 ± 0.7, for 24hGH mean 1.38 ± 1.1, for AUCt 1.38 ± 1.1 and for GH-baseline 1.85 ± 1.3. The GH concentration for all samples in the 24hGH-profile versus amount of non-22kDa GH are shown as polyclonal vs. the ratio polyclonal/monoclonal GH antibodies, see IGFs: Mean IGFISDS, IGFBP3SDS, and the IGFI/IGFBP3 ratioSDS at start were statistically lower in NS than in TS , see SDS correlated positively with short-term changes in IGFISDS and IGFI/IGFBP3-ratioSDS , IGFI/IGFBP3 ratioSDS at start (p=0.033), and chronological age at start of treatment (p=0.037), accounted for 59% of the variance in the first-year growth response. For the TS group, the rhGH dose (p = 0.0091) and the mean 24hGH (p = 0.022) explained 28% of the variance.The main finding of the present study was that NS-children showed higher response in IGFI as well as in growth, pointing to higher responsiveness to GH treatment compared to TS-girls.In the NS-children but not in the TS-children these two GH effects showed great correlation.The magnitude of mean growth response, independent of diagnosis and GH dose, was in parallel with findings in other patients with short stature without GH deficiency –34. GirlSDS -the three selected pre-treatment variables by the multivariable regression models. In girls with TS, the rhGH dose and mean 24hGH became the selected variables explaining only 28% of the variance. The influence of rhGH dose in TS is in accordance with the KIGS outcome research database  and an unrestricted grant from NovoNordisk in Denmark. The authors declare that this study received funding from NovoNordisk. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.Free drugs were given from NovoNordisk for NS and Pharmacia/Pfizer for those TS girls who received the higher rhGHdose. None of the pharmaceutical companies influenced or had any impact on the data analyses nor the writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.